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Type 3 inositol 1,4,5-trisphosphate receptor modulates cell death

SETH BLACKSHAW^*,1, AKIRA SAWA^*,1, ALAN H. SHARP, CHRISTOPHER A. ROSS^*,, SOLOMON H. SNYDER^*,,§2 and ADIL A. KHAN^*

Departments of
^* Neuroscience,
^§ Pharmacology and Molecular Sciences, and
Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA

²Correspondence: *Departments of Neuroscience, ^§Pharmacology and Molecular Sciences, and Psychiatry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA. E-mail: ssnyder{at}jhmi.edu

	ABSTRACT

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Mechanisms accounting for the cellular entry of calcium that mediates cellular proliferation and apoptosis have been obscure. Previously we reported selective augmentation of type 3 inositol (1,4,5) trisphosphate receptors (IP₃R3) in lymphocytes undergoing programmed cell death, which was prevented by antisense constructs to IP₃R3. We now report increases in mRNA and protein levels for IP₃R3 associated with cell death in several apoptotic paradigms in diverse tissues. Elevations of IP₃R3 occur during developmental apoptosis in early postnatal cerebellar granule cells, dorsal root ganglia, embryonic hair follicles, and intestinal villi. Neurotoxic damage elicited by the glutamate agonist kainate is also associated with IP₃R3 augmentation. In chick dorsal root ganglia neurons undergoing apoptosis due to deprivation of nerve growth factor, levels of IP₃R3 are selectively increased and cell death is selectively prevented by antisense oligonucleotides to IP₃R3. Thus, IP₃R3 appears to participate actively in cell death in a diversity of tissues.—Blackshaw, S., Sawa, A., Sharp, A. H., Ross, C. A., Snyder, S. H., Khan, A. A. Type 3 inositol 1,4,5-trisphosphate receptor modulates cell death.

Key Words: nerve growth factor • dorsal root ganglia • PCD • IP₃R3 expression

	INTRODUCTION

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PROGRAMMED CELL DEATH (PCD) is a form of cell death that requires RNA and protein synthesis, in essence actively participating in their own demise (1 2 3) . These features, referred to as apoptosis, include blebbing of the plasma membrane, widespread chromatin condensation, and activation of calcium sensitive endonucleases and proteases. Changes in calcium homeostasis are a frequent feature of apoptosis (4) . Inositol 1,4,5-trisphosphate (IP₃) plays an important role in intracellular calcium signaling and its receptors, IP₃Rs comprise a class of calcium channels with three discrete types derived from distinct genes (5) . In lymphocytes, Ca²⁺-dependent apoptotic death appears to be mediated by type 3 IP₃R (IP₃R3) (6) . IP₃R3 is selectively augmented in lymphocyte apoptosis; IP₃R3 antisense constructs selectively block apoptosis, implying a causal association (6) . PCD is prominent during development of several tissues and after neurotoxic insults (7 8 9 10 11) . We now report augmentation of IP₃R3 expression in several forms of apoptosis. We also provide evidence for the active participation of IP₃R3 in apoptosis, as cell death in dorsal root ganglia neurons is selectively prevented by antisense to IP₃R3.

	MATERIALS AND METHODS

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Chemicals
Unless noted, all chemicals were purchased from Sigma (St. Louis, Mo.). Antibodies against rat IP₃R1 and IP₃R3 were described previously (6) . IP₃R antisense and sense 18 base oligodeoxynucleotides were directed to unique 5' and internal regions of IP₃R1 (bp 321–338 and 1308–1325) and IP₃R3 (bp 126–143 and 2017–2034). Oligodeoxynucleotides were phosphorothioate modified at the first two and last two bases as described before (12) . Antisense oligonucleotides were added to dorsal root ganglia (DRG) cultures every 48 h to maintain a final concentration of 5 µM.

In situ hybridization
In situ hybridization was performed essentially as described (13) . Fresh-frozen, postfixed sections were hybridized with 300 ng/ml unhydrolyzed DIG-labeled probe overnight at 72°C. Sections were washed at 72°C in 0.2xSSC 2 x 1 h and incubated overnight at 4°C in 4% NGS in TBS with antidigoxygenin-AP antibody (Boehringer, Mannheim, Germany) at 1:5000 dilution. Unique probes to IP₃R1, IP₃R2, and IP₃R3 were generated from the 3'UTR of the cDNAs (8571–9335 bp for IP₃R1, 8455–91883 bp for IP₃R2, 8202–8732 bp for IP₃R3). These sequences were generated by polymerase chain reaction, subcloned into pBS, and antisense and sense cRNA probes were generated by T7 and T3 RNA polymerases. Sense control probes used at equal concentration generated no specific signal.

DRG culture
DRG culture was carried out with 9- to 11-day-old chick embryos. DRG were plated on chambers coated with Matrigel matrix (Collaborative Biomedical, Bedford, Mass.). Neurons were cultured 4 to 6 days in Dulbecco’s modified Eagle medium plus 10% fetal bovine serum, penicillin G (100 u/ml), streptomycin (100 mg/ml), cytosine 5-fluro-2-deoxyuridine (10 mg/ml), and nerve growth factor (NGF) 2.5S (100 ng/ml). NGF-containing media was replaced with NGF-free and serum-free media. Neuronal survival was evaluated by counting the number of neuronal cell bodies under a microscope. More than 2000 neurons in 20 randomly selected fields from five independent experiments were counted in the fashion of a blind study.

	RESULTS

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Augmentation of IP₃R3 expression at an mRNA level associated with developmental cellular turnover
Epithelial cells in the villi of the intestine have a high turnover rate in which cells arise from a proliferative zone of undifferentiated stem cells within the crypts, migrate up the villus, differentiate, and are shed apoptotically toward the lumen (14) . IP₃R3 staining is pronounced during embryonic development in intestinal villi (Fig. 1A ). Although the level of IP₃R3 staining is lower in adult rat intestine, it is still confined predominantly to clusters of neighboring villi (Fig. 1A ). By contrast, IP₃R1 staining is most prominent in cells within crypt proliferative zones and decreases in cells as they migrate up the crypt–villus axis, with no difference between embryonic stages and adulthood (Fig. 1A ). IP₃R1 staining is also present in the lamina propria, submucosa, and smooth muscle cells in the muscularis layer, where IP₃R3 is absent (Fig. 1A ).

View larger version (59K): [in this window] [in a new window]   Figure 1. Increases of IP3R3 in rapidly renewing tissues. A) IP3R3 staining in E20 small intestine is localized to villar cells. Black arrows indicate IP3R3 staining localized to villar cells; blue arrows indicate lack of IP3R3 staining in crypt cells, lamina propria, or submucosa. IP3R3 staining in adult gut is localized to groups of villar tips (black arrow). The blue arrow indicates low levels of IP3R3 in crypt cells. IP3R1 staining in E20 small intestine is localized to mitotically active zones in cells of the crypts (blue arrows indicate IP3R1 staining in crypt cells; black arrow indicates low levels of IP3R1 in villar cells). IP3R1 staining in adult gut decreases along the crypt–villus axis (blue arrow indicates IP3R1 staining in proximal portions of villar processes; black arrow indicates lack of IP3R1 staining in villar tips). B) IP3R3 staining is prominent in E20 hair follicle.

In the skin, hair follicles display the highest cellular turnover, especially during embryonic life (15) . At embryonic day 20 (E20), IP₃R3 staining of hair follicle cells is very intense (Fig. 1B ), whereas IP₃R1 staining is much fainter and not concentrated in follicles (data not shown).

Cell death is a widespread event during the development of the nervous system (7 8 9 10) . Cerebellar granule cells undergo apoptosis uniquely between postnatal days 5 and 10 (P5–10) (10) . In P8 cerebellum, IP₃R3 stains prominently in granule cells, but is at low levels in E20 embryonic cerebellum (Fig. 2 ).

View larger version (87K): [in this window] [in a new window]   Figure 2. Increases of IP3R3 in developmental neuronal death. Low/negligible levels of IP3R3 expression are in E20 cerebellum. High levels of IP3R3 expression are in granule cells in the external granular layer (black arrow) in the P8 rat cerebellum.

Increased IP₃R3 in excitotoxic neuronal cell death
The glutamate agonist kainate elicits apoptotic death of distinct neuronal populations (11) . In the adult brain, IP₃R1 is by far the predominant isoform, IP₃R2 is negligible within neurons, and IP₃R3 levels are low/negligible (16) . IP₃R3 levels are low/negligible in the control hippocampus (Fig. 3 ). After kainate treatment, IP₃R3 staining is markedly increased in pyramidal cells of CA1 and even more prominently in the dentate gyrus. IP₃R3 levels are also augmented in the amygdala and in cortical and cerebellar neurons after kainate treatment (data not shown). IP₃R1 and IP₃R2 staining is unchanged in control compared to kainate-treated rats (data not shown). TUNEL staining, an indicator of DNA fragmentation and PCD (14) , is pronounced in the same areas as IP₃R3 (Fig. 3) .

View larger version (116K): [in this window] [in a new window]   Figure 3. Increases of IP3R3 in excitotoxic neuronal death. Sagittal sections of 3-month-old rat hippocampus show negligible IP3R3 expression with no treatment (top left). Increases in IP3R3 levels in adult rat hippocampus are evident 18 h after intraperitoneal (i.p.) kainate (8 mg/kg) in the CA1 and CA3 areas and in the dentate gyrus; top right). The dentate gyrus shows colocalization of purple IP3R3 alkaline phosphatase and rust-colored TUNEL staining (lower left), which is depicted at high magnification in the lower right figure.

Increased IP₃R3 associated with nerve growth factor deprivation
Deprivation of nerve growth factor in chick dorsal root ganglia provides a classic model of neuronal PCD (7) . DRG of non-limb structures are much smaller than DRG associated with limbs because the non-limb DRG undergo massive apoptosis during embryogenesis, peaking at E15 (9) . By E18–20, this process is largely complete. IP₃R3 staining is pronounced in E15 DRG (Fig. 4A-1 ). We prepared primary cultures from embryonic chick DRG as an experimental system. Cultured DRG cell survival is dependent on NGF, as its deprivation causes DRG cell death (17) . Western blot analysis from cultured cells reveals an augmentation of IP₃R3 protein after NGF deprivation while a modest decrease in IP₃R1 occurs, resembling our findings in apoptotic lymphocytes (6) (Fig. 4A-2 ).

View larger version (30K): [in this window] [in a new window]   Figure 4. IP3R3 antisense oligonucleotides block NGF deprivation-induced neuronal death. A) (1) High-power magnification of IP3R3 expression in developing dorsal root ganglion at E15. (2) Protein immunoblot detection of IP3R with affinity-purified, subtype-specific rabbit anti-IP3R1 and IP3R3 antibodies. Lane NGF +: control NGF-treated DRG homogenates. Lane NGF -: NGF-deprived DRG homogenates. Molecular mass is shown at the left (in kilodaltons). Results are from a representative one of four experiments that produced the same results. (3) Protein immunoblot detection of IP3R3 in DRG neurons incubated with sense IP3R3 oligonucleotides (lane 3S), antisense IP3R3 oligonucleotides (lane 3AS), and antisense IP3R1 oligonucleotides (lane 1AS) 7 days after NGF deprivation. Molecular mass shown at the left (in kilodaltons). The experiment was replicated four times with closely similar results. B) Left: Phase contrast photomicrographs of DRG neurons. DRG neurons were grown in vitro in NGF for 5 days, switched to a NGF-free serum-free media for 10 days, and cocultured with antisense IP3R3, antisense IP3R1, sense IP3R3, and sense IP3R1 oligonucleotides, respectively. DRG cell viability without NGF was enhanced with antisense oligonucleotides to IP3R3. Panel NGF (+): DRG neurons treated with NGF for 10 days; NGF (-) 3S: DRG neurons, 10 days after NGF deprivation, cocultured with IP3R3 sense oligonucleotides; NGF (-) 3AS: DRG neurons, 10 days after NGF deprivation, cocultured with IP3R3 antisense oligonucleotides. Right: Viability was evaluated as the number of neuronal cell bodies per field. More than 300 neurons were counted for each field examined. Data are presented as means ± SE for independent experiments. Viability in samples treated with IP3R3 antisense is significantly greater than untreated samples or samples treated with IP3R3 sense, IP1R3 3AS antisense, or scrambled oligonucleotides (P<0.005). 3AS: antisense to IP3R3; 3S: sense to IP3R; 1AS: antisense to IP1R3; SC: scrambled oligonucleotides.

Antisense to IP₃R3 prevents cell death in dorsal root ganglia neurons after nerve growth factor withdrawal
In lymphocytes, we showed that IP₃R3 mediates apoptosis by establishing stable cell lines expressing IP₃R3 antisense, which rescued the cells from apoptotic death, but IP₃R1 antisense constructs were ineffective (6) . In chick DRG, we have used antisense oligonucleotides. Western blot analyses show that treatment with IP₃R3 antisense reduces the increase of IP₃R3 protein associated with NGF deprivation (Fig. 4A-3 ). No such effect occurs with IP₃R3 sense, or IP₃R1 antisense treatment.

In control DRG, NGF deprivation elicits blebbing, fragmentation of neuronal cells and fibers, and the accumulation of debris and reduces viability to 20–25% of values in the presence of NGF (Fig. 4B ). IP₃R3 antisense treatment restores viability to 68% of values for cultures containing NGF. DRG treated with IP₃R1 antisense, IP₃R3 sense, or scrambled probes appear the same as DRG-deprived of NGF with extensive cell death (Fig. 4B ).

	DISCUSSION

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In an earlier study we provided evidence for a causal role of IP₃R3 in eliciting cell death in lymphocytes (6) . In that study we showed an augmentation of IP₃R3 but not IP₃R1 or IP₃R2 associated with PCD. Moreover, stable expression of antisense to IP₃R3 prevented apoptotic death, whereas transfection of IP₃R1 antisense constructs was ineffective. The present study was undertaken to ascertain whether the link of IP₃R3 and PCD is restricted to lymphocytes. Models of developmental cell death, including cellular turnover associated with the development of intestinal cells, hair follicles, and cerebellar granular cells, are all associated with selective increases in IP₃R3. Similarly, IP₃R3 is augmented selectively after excitotoxic neuronal cell death in the hippocampus, as well as after deprivation of nerve growth factor from DRG.

In the DRG system, we show an apparent causal link of IP₃R3 to cell death, as antisense to IP₃R3 (but not to IP₃R1) prevents cell death. Thus, IP₃R3 appears associated with PCD in multiple cellular models; in two of these, antisense constructs prevent death.

After our initial observations in lymphocytes (6) , several groups have also reported involvement of IP₃Rs in PCD especially in various types of lymphocyte preparations. Jayaraman and Marks (18) studied PCD after several types of insults to Jurkat T lymphocytes. They were able to prevent PCD by stably transfecting a full-length antisense construct modeled on IP₃R1. However, because a full-length construct was used and because IP₃R1 and IP₃R3 have extensive sequence homology, the constructs they used may have influenced both subtypes of IP₃R. In B cells Sugawara et al. (19) found that deletion of any two IP₃R subtypes or all three isoforms together reduced cell death following activation of B cell receptors, whereas deletion of individual IP₃R subtypes was not effective. The study by Sugawara is different from that of Khan et al. (6) in that the former group stimulated PCD by B cell receptor activation and Khan et al. used treatment with dexamethasone. In prostate tissue, IP₃R3 appears to be selectively associated with PCD, as antisense constructs to IP₃R3 (but not to IP₁R3) prevent PCD (B. Tombal, A. Sawa, S. H. Snyder, and J. T. Isaacs, unpublished results). Thus, several laboratories have observed an association of IP₃R with various models of PCD. Some workers have provided evidence for participation of multiple subtypes of IP₃R, which is in contrast to the selective role of IP₃R3 we have observed. It is unclear whether these differences are related to variations in the stimuli used to elicit PCD or to tissue-specific variations.

Cellular proliferation, though opposed in function to apoptosis, also requires calcium, and proliferative stimuli elicit calcium release attributed to IP₃ (20) . Our preliminary results suggest that IP₃R1 but not IP₃R3 plays a role in the initiation of cellular proliferation, as peripheral lymphocytes stimulated with concanavalin A displayed a fourfold increase in IP₃R1 mRNA with no increase in IP₃R3 (A. A. Khan, and S. H. Snyder, unpublished data).

A role for IP₃Rs in PCD presumably involves release of intracellular calcium, which is thought to be the principal function of IP₃Rs. Several lines of evidence suggest that differential expression of IP₃R subtypes alters the spatial and temporal pattern of the calcium signal. Differential immunohistochemical localizations of IP₃R associated with endoplasmicreticulum, nuclear membrane, or plasma membrane may vary with receptor subtypes (6 , 21) . Overexpression of IP₃R3 in Xenopus oocytes augments the magnitude and duration of calcium influx with IP₃R3 concentrated near the plasma membrane, whereas overexpression of IP₃R1 enhances intracellular calcium release (22) . Different temporal patterns of calcium signals in chicken B cells depend on the subtype of IP₃Rs (23) .

Extensive studies have been devoted to clarifying ways in which calcium is associated with PCD. Calcium-activated proteases such as calpain are implicated in PCD (24) . Activation of the calcium-dependent phosphatase calcineurin is also linked to PCD (25 , 26) . Increased cytosolic calcium is sequestered by mitochondria and can cause mitochondrial membrane depolarization, which is an initial event in PCD (27 28 29) . Abnormalities of mitochondrial polarity have been observed in various toxic paradigms including PCD elicited by dexamethasome, irradiation (28) , and in disease models such as Huntington’s disease, in which lymphocytes of the patients display marked sensitivity to mitochondrial depolarization (30) .

	ACKNOWLEDGMENTS

 
We thank Dr. John Isaacs for helpful discussions. We thank D. Dodson for typing the manuscript. We thank C. Williams and A. M. Kodaira for technical assistance. Supported by USPHS grant MH-18501 and Research Scientist Award DA-00074 to S.H.S.

	FOOTNOTES

 
¹ These two authors contributed equally to this work.

Received for publication September 24, 1999. Accepted for publication November 11, 1999.

	REFERENCES

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